Natural killer (NK) cells play an important role in host defense against tumors and infectious agents. They induce target cell death, primarily by the release of cytotoxic granules containing perforin and granzymes. In studies to characterize additional proteins associated with cytolysis, a novel protein whose expression was highly increased upon cytokine stimulation of NK cells was identified. This protein was named NK lytic associated molecule (NKLAM) and is the subject of this ongoing VA merit grant project. NKLAM is a RING finger transmembrane protein localized to NK cytolytic granule membranes. Studies have shown a role for NKLAM in NK-mediated killing of tumor cells. NKLAM is also up-regulated in macrophages upon toll-like receptor (TLR) stimulation, suggesting a role in bacterial killing as well. To further assess the role of NKLAM, NKLAM deficient (KO) mice were generated. These mice exhibit 50% less NK activity than wild type (WT) mice and produce less interferon-? in response to tumor cell contact. NKLAM KO mice also have substantially higher numbers of lung metastases compared with WT after injection with B16 melanoma cells and show greater dissemination of lymphoma cells to lymph nodes from the primary tumor site. Finally, a critical finding is that NKLAM functions as an E3 ubiquitin ligase. Ubiquitination is a key mechanism for regulating immune responses. In vitro studies of NKLAM KO and WT NK cells and macrophages suggest that NKLAM participates in cytokine and TLR-mediated signaling events. In this model, NKLAM regulates NK/macrophage activity by modulating signaling events in effector cells. Alternatively, preliminary studies have shown that NKLAM is released into the supernatants of NK cells upon degranulation of effector cells. Accordingly, another way NKLAM may function is to be transported into the target cell after release by the effector cell. In this model, NKLAM, acting as an E3 ligase, would ubiquitinate anti-apoptotic or growth associated molecules in the target, resulting in their degradation, thereby promoting target cell apoptosis. We identified a protein, uridine-cytidine kinase-like 1 (UCKL-1), which is ubiquitinated by NKLAM. The function of this novel protein is unknown. However, its homology to uridine kinases and over-expression in tumor cells suggests a role for UCKL-1 in tumor growth and/or survival. To test this, RNA interference (RNAi) was used to down-regulate UCKL-1 expression in NK-sensitive K562 erythroleukemia cells and other NK targets. Decreased expression of UCKL-1 in K562 slows their proliferation, induces apoptosis and enhances their susceptibility to NK-mediated lysis; over-expression of UCKL-1 reduces their susceptibility to NK lysis. Preliminary studies show that NKLAM may act at the level of the effector cell and/or target cell. These two models are not mutually exclusive and may both be functional depending upon the circumstances. This application consists of three independent but interlinked aims to study the E3 ligase function of NKLAM and its functional consequences in NK cells, macrophages and target cells. 1) Determine the role of NKLAM in NK cells and target cells. The role of NKLAM in signaling and cytotoxic function in NK cells will be assessed. Studies will also be performed to test the hypothesis that upon NK cell degranulation, NKLAM enters target cells and ubiquitinates proteins such as UCKL-1, enhancing target cell death. Studies are proposed to further elucidate the function of the novel protein UCKL-1 in vitro and in vivo. 2) Determine the role of NKLAM in macrophages. The role of NKLAM in TLR-mediated signaling events and in macrophage phagocytosis will be investigated. 3) Characterize NKLAM KO mice to study the role of NKLAM in vivo. A variety of tumor models will be evaluated to determine where NKLAM plays a role. Analysis of NKLAM KO mice may reveal additional roles for NKLAM and potentially unveil alternative mechanisms of cell killing. PUBLIC HEALTH RELEVANCE: The long term goal of these studies is to investigate how the anti-tumor and anti-microbial activities of NK cells and macrophages are regulated and how they can be enhanced. NKLAM is an important component of these immunological processes. Understanding the normal mechanisms of anti-tumor and anti-bacterial immunity is a critical first step in developing new therapies, and ultimately, preventing cancer and infectious disease. These diseases are of great relevance to the VA health care system. Our military personnel throughout the world are being exposed to new, resistant microbial pathogens that have emerged and are spreading. They may also be in contact with toxic, cancer causing agents. As our veteran population is aging, deficiencies in immunological functions are associated with increased tumor incidence and increased susceptibility to infection. The proposed studies have the potential to lead to new strategies for disease prevention or treatment.